Detection of mycobacteria

Mycobacteria are usually detected by auramine fluorescent staining, and the results are available within 1 working day. Laboratories may also offer molecular amplification tests for the detection of Mycobacterium tuberculosis in smear positive pulmonary specimens and in possible cases of tuberculous meningitis. Two specimens of cerebrospinal fluid (CSF) are usually needed to enhance sensitivity.

In all cases, the specimens are usually processed using rapid culture systems - for example, MB/BacT or BacT/ALERT/3D or MGIT. Molecular amplification techniques, such as polymerase chain reaction (PCR), are less sensitive than culture but faster; in numerous studies, PCR-based detection systems using various gene targets have detected 90% to 95% of smear positive, culture positive isolates, but only between 60% to 70% of all smear negative, culture positive isolates.

Isolation of mycobacteria

Mycobacteria should be isolated by rapid automated liquid culture and by conventional solid culture. Local laboratories that handle few specimens should consider referring primary specimens to a reference laboratory for mycobacterial culture. All reference laboratories are able to advise on and perform rapid culture. Specimens can be referred to the reference laboratories as original specimens. The processing time will depend on the species and smear positivity of the specimen.

M. tuberculosis is usually detected and identified within 7 to 21 days depending upon biomass, which is also reflected in the smear result. Positive cultures are reported as they are detected and all negative cultures will normally be incubated for at least eight weeks before a negative report is sent out. When unusual mycobacterial species are suspected then incubation will be further prolonged.

Identification of mycobacteria

Isolates may be submitted to the reference laboratories by courier as culture slopes or as growths in liquid medium. Such cultures are assigned as UN 3373 (Diagnostic specimens) and referring laboratories should ensure that all specimens are packed and marked according to Packing Instructions P650. In particular, mycobacterial cultures should be sent in screw-top, preferably plastic or thick-walled glass, bijou or universal bottles packed inside a rigid container with enough absorbent material to absorb the contents if the bottle breaks.

Identification normally includes molecular DNA analysis and/or standard numerical taxonomy procedures based on macroscopic and microscopic appearances, growth, biochemical characteristics and drug resistance. All reference laboratories report receipt of the isolate and initial identification results to the source laboratory within 1 working day.

Susceptibility testing of clinically relevant isolates

Drug susceptibility testing has 3 main goals:

to facilitate the management of individual patients, particularly if drug resistance is likely

to provide data on which to plan drug combinations for treatment

to provide a surrogate measure of the relative effectiveness of tuberculosis control programmes

Drug susceptibility testing is performed using one of three main methods: absolute concentration method, the resistance ratio method, or the proportion method. All of these methods are acceptable if their quality is adequately controlled and validated. All reference laboratories employ the resistance ratio method using solid media and/or the proportional method using the automated liquid culture systems. Results of tests for primary therapeutic agents should be completed within 14 days of receipt of the isolate by the reference laboratory. However, for some atypical mycobacteria, this may be longer.

All reference laboratories perform drug susceptibility testing for first line and (with the exception of Northern Ireland) most second line chemotherapeutic agents. Susceptibility testing for second line drugs is performed whenever resistance to more than one first line drug is identified, if the patient has been in contact with drug resistant cases, or if multi-drug resistant tuberculosis is more likely (for example, in patients who are poorly compliant with treatment, have had treatment for a prior infection, or on request, such as when a patient has impaired absorption with or without vomiting). Molecular assays are also available to detect rifampicin resistance from cultures and primary specimens.

The NMRL performs tests for third line drugs such as ethionamide, cycloserine, capreomycin and para-aminosalicylic acid (PAS); other agents may be tested as needed. The NMRL is also a WHO SupraNational Reference Laboratory and European Co-ordinating Centre within the Global Programme on Drug Resistance and operates an EQA scheme for drug resistance on behalf of the WHO.

Atypical mycobacteria are commonly found in the environment and their clinical significance depends on the body site from which the organism is isolated and the frequency of isolation. The likely clinical significance is a criterion used to decide whether drug susceptibility tests will be performed. Clinical details must therefore accompany the specimen as results can be interpreted only in clinical context.

Help us improve GOV.UK

Help us improve GOV.UK

To help us improve GOV.UK, we’d like to know more about your visit today. We’ll send you a link to a feedback form. It will take only 2 minutes to fill in. Don’t worry we won’t send you spam or share your email address with anyone.